Regeneration

ABSTRACT

The invention relates to the field of regeneration of cells and the vegetative propagation of (micro)-organisms or specific parts such as tissues or organs thereof, for example of those cells grown in tissue or organ culture, and more in particular to the seedless propagation of plants. The invention provides a culture method for propagation of a plant from plant starting material wherein during regeneration of said starting material, especially in the phase of the development of the shoot-root body plan, root or shoot initiation is stimulated by a recombinant gene product or functional fragment thereof, for example derived from a gene involved in the regulation of plant development allowing reducing or omitting exogenous phytohormone addition to said culture.

The invention relates to the field of regeneration of cells, self-renewal of (micro)-organisms, the vegetative propagation of plant parts such as plant tissues or organs thereof, for example cells grown in tissue or organ culture, and more in particular to the seedless propagation of plants.

Renewal of plant and animal cells into more cells, tissues, organs and even whole plants and organisms is a process central to life that has been set to men's whims and desires already for a long time. Self-renewal of specific micro-organism starter cultures are used to ferment foods and drinks. Yet other cultures are useful for the metabolites they produce per se, such as produced by modern day's large scale fermentor cultures for the production of antibiotics or enzymes. Within the realm of animal cells, use of the renewed cultured cells, although being of fairly recent date, has taken great flight with the production of for example viral vaccines in cell- or tissue culture. Even more recent is the use of donor cells harvested from an individual, and grown and/or differentiated in culture, for transplantation purposes. Such cells (take for example bone marrow cells) are, after having been sufficiently regenerated and differentiated, proliferated or equipped with the desired characteristics, transplanted into a recipient for medical purposes. Shortly, such therapies will even include transgenic cells, transformed with modern recombinant techniques, that are thereby equipped with the desired characteristics and transplanted.

Regeneration is very well studied in plants, where it is crucial in vegetative propagation. In principle, plants can be propagated in two ways, via seeds or vegetatively without using seeds as starting material to obtain the desired plant. Both types of propagation may be impossible or undesirable under certain conditions. When propagation via seeds is unsatisfactory (when no seeds or too few of the desired seeds are formed or the desired seeds quickly loose their germination viability) then seedless propagation is often adopted. Also, when due to sexually crossing a very heterogenous progeny is or may be obtained due to its strong heterozygosity, propagation via seeds is often also considered unsatisfactory. Of course, seedless propagation of essentially seedless starting material may in a later phase give rise to the desired seeds, which can further be used to obtain the desired plants.

Within seedless propagation of plants two major fields can be distinguished: In vivo and in vitro vegetative propagation. In vivo vegetative propagation (via for example cuttings, splitting or division, layering, earthing up, grafting or budding, and other methods known to the gardener or horticulturist), has for many years played an important role in agriculture; e.g. with potatoes, apples, pears, many ornamental bulbs and tuberous plants like potatoes, many arboricultural crops, carnations, chrysanthemums, etc. Vegetative propagation is also very important in plant breeding: parent lines have to be maintained and propagated vegetatively for seed production; cloning is often required for setting up gene banks; adventitious shoot formation is needed to obtain solid mutants after mutation induction.

However, the classical methods of in vivo vegetative propagation often fall short (to slow, too difficult or too expensive) of that required or are completely impossible. In the last couple of decades, since the discovery that plants can be more rapidly cloned in vitro than in vivo, knowledge concerning vegetative propagation has grown quickly; this holds equally true for plants from temperate, subtropical as well as tropical regions. It has now even become possible to clone species by in vitro culture techniques that are impossible to clone in vivo. Different methods of in vitro vegetative or seedless propagation from plant starting material are for example using single-node cuttings, axillary branching, regeneration of adventitious organs (roots or shoots) on starting material such as explants or callus tissue and regeneration of plants from suspensions of, or even single, cells or protoplasts used as starting material. For the generation of transformed or transgenic plants, in vitro propagation is even considered a prerequisite, since it is the totipotency of individual plant cells that underlies most plant transformation systems.

To propagate plants from starting material in vitro, it is in principle necessary that at least one cell in the starting material is capable of regeneration. The ability to regenerate is for example determined by the genotype, the environmental conditions (nutrient supply, regulators and physical conditions) or the developmental stage of the plant, or combinations of these. It is well known that some families and genera have high regeneration ability: Solanacea (Solanum, Nicotiana, Petunia, Datura, and Lycopersion), Crucifera (Lunaria, Brassica, Arabidopsis), Generiaceae (Achimenes, Saintpaulia, Streptocarpus) Compositae (Chicorium, Lactuca, Chrysantemum), Liliaceae (Lilium, Haworthia) Allium, Ornithogalum) but others, such as many decorative plants, woody species such as shrubs, conifers or trees, especially fruit trees, Rosacea, Alstroemeria, Euphorbia, and bulbs such as Tulipa, and others are notoriously difficult, even with in vitro techniques.

As indicated above, regeneration (self-renewal of (micro-)organisms and self-renewal of plants, animals or parts thereof, i.e. vegetative reproduction/propagation) can also be considered a repair strategy observed throughout the realm of micro-organisms, animal and plant species. Regeneration in plants for example comprises the formation of new tissues containing both root and shoot meristems, separate shoot or root meristems, plant organs or organ primordia from individual cells or groups of cells. Regeneration in general mimics the process of normal cellular and organ differentiation that takes place during plant development and results in the formation of the different plant organs. In normal development, early in ontogony, cells and tissues of common lineage diverge into often contrasting paths of development as they respond to developmental signals. This ability to develop in response to a specific signal is also known as cellular competence or cellular potentiality. As competent cells become committed to particular paths of differentiation, they are not readily diverted into other pathways; this restriction of the developmental potentiality of cells is referred to as determination.

Plant cells or groups of cells that under normal conditions are unable to initiate the formation of certain plant organs, meristems or organ primordia can often be stimulated by extracellular stimuli modifying the differentiation stage of the cell. Extracellular diffusible factors have shown to be essential for cellular redifferentiation in plant cells (Siegel and Verbeke, 1989 Science 244, 580-582). The perception of these signals at the cellular surface and the intracellular signal transduction that finally result in changes in transcriptional regulation provides cells with the ability to respond to such extracellular stimuli. Regeneration can result in the formation of either a shoot alone or a root alone or both together. Only after redifferentiation of a cell or tissue, regeneration is possible that results in differentiated tissue that again comprises the necessary three-dimensional layout of the emerging plant, the apical-basal or shoot-root body plan from which the mature desired plant can develop.

Indeed, central in in vitro techniques for seedless propagation are phytohormones and other factors often added to the culture medium that mimic these extracellular stimuli. For the process of regeneration of the original starting cell into a multicellular totipotent tissue underlying and preceding somatic embryogenesis or organogenesis in vitro in cell, tissue or explant cultures which lead to a fully differentiated plant again, in general a well balanced, and per plant species often different, phytohormone addition to the culture is required. Overall, a balance is required between auxins on the one hand and cytokinin on the other. After exogenous exposure to auxin (such as 2,4-dichlorophenoxyacetic acid (2,4-D), chloramben or dicamba) or cytokinin (such as 6-benzylaminopurine or zeatine) or both, cells or tissue react by development of the shoot-root body plan, for example by forming shoots and/or roots, sometimes readily, sometimes erratically especially when the proper balance between the hormones is not properly selected.

Regeneration in vitro and especially the manipulatable nature of in vitro culture thus depends mainly on the application of these two types of hormones, and also on the ability of the tissue to respond to phytohormonal changes during culture. In general, three phases of regeneration are recognisable. In the first phase, cells in the culture acquire “competence”, which is defined as the ability (not capacity) to respond to hormonal signals of organ induction. The process of acquisition of said organogenic competence is often referred to as “dedifferentiation” of differentiated cells to acquire organogenic competence. The competent cells in the culture are canalised and determined for specific tissue and organ formation for re-entry of quiescent cells into cell cycle, and organisation of cell division along the lines of the shoot-root body plan to form specific primordia and meristems under the influence of the phytohormone balance through the second phase. Especially auxin is thought to be involved in specific regenerative signal transduction pathways for adventitious root initiation, whereas cytokinin is thought to be involved in specific regenerative signal transduction pathways for adventitious shoot initiation.

Then the morphogenesis, the growing of the plant to its fully differentiated state, proceeds independently of the exogenously supplied hormones during the third phase.

Although the general principles governing regeneration via addition of exogenous phytohormones are thus fairly well understood, designing working in vitro culture protocols finding the right balance, the right time of administration or the right type or subtype of said hormones for a great many individual species is still more or less a process of trial-and-error. However, as already indicated above, for in vitro regeneration or seedless propagation of a great many plant species is a large interest, especially for those that are in general hard to propagate.

The invention provides a culture method for propagation of a plant from plant starting material wherein, especially in the phase of the development of the shoot-root body plan, root or shoot initiation is stimulated by introducing at least one recombinant gene product or functional fragment thereof in said starting material, for example by stimulating at least one signal transduction pathway for root or shoot initiation, said gene product or gene products for example derived from a gene or genes involved in the regulation of plant development, allowing reducing or omitting exogenous phytohormone addition to said culture in the regeneration process. In a preferred embodiment the invention provides a culture method for vegetative propagation of plants from plant starting material comprising regeneration of said starting material wherein during regeneration of said starting material at least one specific signal transduction pathway for adventitious root or shoot initiation is endogenously stimulated allowing reducing or omitting exogenous phytohormone addition to said culture, in particular wherein said pathway is endogenously stimulated by a recombinant gene product derived from a gene involved in the developmental regulation of regeneration, such as a gene or gene product involved in hormone production, a gene or gene product giving feed back on hormone production, or involved in the cascade of events leading to regeneration.

Preferably, the method as provided by the invention comprises at least one step of in vitro culture, since it is in in vitro culture that the auxins or cytokinins are most widely used, in the regeneration process, especially for plants that are notoriously difficult to regenerate for vegetative propagation such as many decorative plants, woody species such as shrubs, conifers or trees, especially fruit trees, Rosacea, Alstroemeria, Euphorbia, and bulbs such as Tulipa. However, clearly, said hormones are also commonly used in in vivo cultures as well, (in vivo cultures essentially being all crop or plant culture methods traditionally used in agriculture) where such hormones are commonly added by (root or stem) dipping, spraying or watering. Especially those plants that are propagated in an essential seedless way can now be regenerated or propagated more easily, consequently, in a preferred embodiment, the invention provides a culture method for essentially seedless propagation of plants from plant starting material comprising regeneration of said starting material wherein during regeneration at least one specific signal transduction pathway for adventitious root or shoot initiation endogenously is stimulated, e.g. by above mentioned gene product, allowing reducing or omitting exogenous phytohormone addition to said culture.

Essentially seedless propagation herein is defined in that said starting material essentially comprises no seeds, or at least that seed possibly present in said starting material does not lay at the basis of the regeneration of said starting material or does not develop into the desired plant. However, as one aspect of the culture method comprising regeneration as provided by the invention, during or after the process of regeneration or propagation according to the invention seed may be formed, from which even a desired plant may develop, which is a result of the propagation according to the invention, rather than that it lays at the basis thereof.

In particular, the invention provides a culture method wherein said starting material comprises an individual plant cell or protoplast or explant or plant tissue, materials which are commonly used in in vitro culture methods whereby the addition of phytohormones was thought to be axiomatic. Now such addition is no longer necessary or can be reduced, providing an easier way of in vitro culture, wherein not such an intricate balance between the addition of the various hormones has to be sought.

The invention provides manipulation of propagation characteristics of for example plant tissue. Numerous plant species are propagated in tissue culture in order to obtain large amounts in a relative short period of time. Using the invention it is relatively easy to increase the multiplication factor several times. For several notoriously difficult species, like shrubs, trees en various bulbous species it is now also possible to use essentially seedless propagation, and especially in vitro culture, when using the invention. The regeneration capacity of cells or tissue isolated from these plants is increased significantly, thereby increasing the multiplication factor by introducing of certain bioactive molecules, like nucleic acid or (modified) protein. The nucleic acids or proteins may be introduced by the methods known in art, like particle gun bombardment, electroporation, micro-injection or other techniques described in the introduction. The introduced molecules are either nucleic acid, being RNA, or naked DNA with a small chance of becoming integrated in the genome, or (modified) protein product. The molecules will in general be lost during the regeneration process and are therefore only transiently present. The nucleic acids that may be used encode or produce proteins that stimulate the regeneration process and reduce or eliminate the use of exogenously added planthormones. The proteins that may be added are the protein products of these nucleic acids or their modified forms. Examples of molecules with the above described characteristics are proteins or genes coding for proteins involved in the regulation of plant development or perception of plant hormones. By using the invention the multiplication factor can be increased so much that it will be possible to use in vitro propagation techniques in a broader sense and also for the more difficult species, Also, by using the invention it is relatively easy to permanently increase the propagation characteristics for these plants. The regeneration capacity of these plants can be increased significantly if these plants are made transgenic by introducing a gene coding for proteins involved in the regulation of plant development or perception of plant hormones or more specific a gene coding for a product stimulating or inducing one signal transduction pathway for root or shoot initiation or even more specific a gene coding for a representative of the plant receptor kinase family RKS. Transformation can be achieved using the techniques known in the field like Agrobacterium mediated transformation, particle gun bombardment, the above described marker-free transformation system or others and select for non-lethal expressors of the gene.

In one preferred embodiment, the invention provides a culture method according to the invention wherein said starting material comprises a desired somatic mutation. Mutations can occur in any cell of a living organism, but are only transferred to the offspring when this mutation occurred in those cells from which gametophytic cells of that organism are derived. Somatic mutations are usually lost unless the tissue in which the mutation is apparent is vegetatively propagated or if cells in this tissue are regenerated to form an intact new organism. Using the technology described in this invention the rescue of somatic mutations in plants is provided. Somatic, but also generative tissue is stimulated to regenerate by the introduction of bioactive molecules, like nucleic acid or (modified) protein as provided by the invention. The nucleic acids or proteins may be introduced by the methods known in art, like particle gun bombardment, electroporation, micro-injection or other techniques described. The introduced molecules are either nucleic acid, being RNA, or naked DNA with a (not necessarily) small chance of becoming integrated in the genome, or (modified) protein product. The molecules will in general be lost during the regeneration process and are therefore in general only transiently present. The nucleic acids that may be used encode proteins that stimulate the regeneration process and reduce or eliminate the use of exogenously added planthormones. The proteins that may be added are the protein products of these nucleic acids or their modified forms. Examples of molecules with the above described characteristics are proteins or genes coding for proteins involved in the regulation of plant development or perception of plant hormones. Alternatively somatic mutations may have been created by treatment of seeds with mutagenic agents, like colchicines, EMS, radiation or carcinogenic substances etc. The sectors in these mosaic plants grown from these treated seeds will be screened for desirable phenotypes. The interesting sectors will subsequently be isolated and used as starting material for regeneration by the above-described invention in order to obtain clonal propagation of these desired traits.

In another preferred embodiment, the invention provides a culture method according to the invention wherein said starting material comprises transgenic material. These days transgenic plants are being produced rapidly, albeit often in only limited numbers. To rapidly acquire sufficient numbers of plants for further propagation under field conditions, in vitro culture techniques are widely used. The invention now provides a method wherein little or no attention has to be given to phytohormone levels in such transgenic plants cultures.

In particular, the invention provided a method wherein said starting material additionally comprises starting material comprising a recombinant nucleic acid encoding a desired trait. The invention herewith provides essentially marker-free transformation, or at least it provides plants that after transformation and propagation are essentially marker-free. A recombinant nucleic acid encoding a desired trait, that one would like to integrate in a plant's genome is provided to at least part of said starting material with gene delivery vehicles or methods, such as vectors, particle bombardment, electroporation, micro-injection or other techniques described in the art. Cells comprising said recombinant nucleic acid are also provided according to the invention with at least one recombinant gene product or functional fragment thereof, for example by stimulating at least one signal transduction pathway for root or shoot initiation, said gene product or gene products for example derived from a gene or genes involved in the regulation of plant development, allowing reducing or omitting exogenous phytohormone addition to said culture. In particular, the invention provides a culture method for vegetative propagation of plants from plant starting material having been provided with a recombinant nucleic acid encoding a desired trait comprising regeneration of said starting material wherein during regeneration of said starting material at least one specific signal transduction pathway for adventitious root or shoot initiation is endogenously stimulated allowing reducing or omitting exogenous phytohormone addition to said culture, in particular wherein said pathway is endogenously stimulated by a recombinant gene product derived from a gene involved in the developmental regulation of regeneration, such as a gene or gene product involved in hormone production, a gene or gene product giving feed back on hormone production, or involved in the cascade of events leading to regeneration.

In a preferred embodiment, said recombinant nucleic acid encoding a desired trait has additionally been provided with means for nuclear targeting and/or integration in a plant genome. Such means can be nucleic acid signals incorporated with the recombinant nucleic acid encoding the desired trait, or proteinaceous substances such as transposases, or viral or bacterial proteins (such as Vir-proteins) to protect the recombinant nucleic acid inside the cell, taking care of proper targeting towards the nucleus and/or stimulating proper integration.

Even more preferred, the invention provides a method wherein said starting material comprises a to be transformed individual plant cell or protoplast or explant or plant tissue comprising recombinant nucleic acid encoding a desired trait among other, non-transformed starting material from which the transformed material has to be selected.

In general, as a part of the process of for example plant transformation, dominant selectable markers are used to select transgenic cells from which transgenic plants can be regenerated. For one thing, these marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, selectable marker genes conferring for example antibiotic or herbicide resistance, used to introduce economically valuable genes into crop plants have major problems: detoxification of the selective agent by expression of a modifying enzyme can enable untransformed cells to escape, dying untransformed cells release products which are toxic and inhibit the regeneration of transformed cells, the selective agents may have negative effects on proliferation and differentiation of cells, there is uncertainty regarding the environmental impact of many selectable genes, and it is difficult to perform recurrent transformations using the same selectable marker to pyramid desirable genes. The invention now provides a method reducing or omitting selective agent addition to said culture.

Attempts have been made earlier to design transformation systems allowing marker gene elimination to obtain marker-free transformants of diverse plant species whereby the marker gene used is removed from the transformed cell after it has performed its task. One method involves co-transformation of cells mediated by Agrobacterium tumefaciens with binary vectors carrying two separate T-DNAs, one for example comprising a drug-resistance selection marker gene, another comprising the desired gene, followed by conventional out-breeding the undesired drug-resistance gene, that is thought to localise at a different locus than the desired gene. Although drug sensitive transformants comprising the desired gene may be thus obtained it is not clear whether all these transformants are indeed totally free of (non or partly functional) selection marker-gene or fragments thereof. Also, the selective agent initially used still has the unwanted negative effects on proliferation and differentiation of plant cell during the transformation process. Furthermore, the method requires sexual crossing which limits it to plant species where sexual crossing, and not vegetative reproduction, is the practical method of reproduction, and practically limits it even further to those plant species with a sufficient short generation time.

One strategy currently available to eliminate the superfluous marker after the cell has been transformed without the need to sexually cross plants is the MAT vector system. However, said system relies on intrinsic post-transformational excision of the selection gene which is comprised in a transposable element, an event which only haphazardly occurs and reduces the final efficiency of the transformation process.

Yet another strategy involves site specific recombination such as seen with the Cre-Lox system whereby in a first transformation the selection-marker gene is inserted at a previously determined specific site, allowing selection of transformed cells, after which in a second transformation comprising the introduction of a site specific recombinase, the selection-marker gene is again excised from the genome.

Needles to say that, apart from other problems, the prerequisite of having a suitable site in the to be transformed cell available restricts said method to those organisms of which the genome is well known. The invention now provides a method to obtain transformed plants by in vitro culture wherein said transgenic material is devoid of a selectable marker gene conferring resistance to an selective agent. Resistance to selective agents is no longer needed since according to the invention the transformed material is equipped with the necessary recombinant gene product or gene products or functional fragment(s) thereof derived from a gene involved in the regulation of plant development allowing reducing or omitting exogenous phytohormone addition to said culture, thereby giving preferred growth conditions to the transformed cells over those non-transformed cells that have not been provided with said gene product or functional fragment thereof. In particular, the invention provides a culture method for vegetative propagation of plants from transformed plant starting material comprising regeneration of said starting material wherein during regeneration of said transformed starting material at least one specific signal transduction pathway for adventitious root or shoot initiation is endogenously stimulated allowing reducing or omitting exogenous phytohormone addition to said culture, in particular wherein said pathway is endogenously stimulated by a recombinant gene product derived from a gene involved in the developmental regulation of regeneration. The beauty of it is that no selectable marker gene conferring resistance to a selective agent has to be introduced in said material at all, thereby obviating the need to deplete the transformed material of such marker genes afterwards. In particular, the invention thus does not make use of resistance to antibiotic or herbicides, and does nor carry all the disadvantages associated herewith.

In short, most plant transformation systems are based on the selection for herbicide or antibiotic resistance or selection for transformants is based on the presence of an additional selection marker besides the trait itself. Using the technology described in this invention, markerless transformation in plants is provided. This new transformation/regeneration (t/r) system for example consist of two components (FIG. 20). A first component in this example is the trait, which may be present between the borders of Agrobacterial T-DNA, but apart from a suitable promoter no other DNA is needed. This first component may be single or double stranded DNA and may be in vitro coated with the VirE2 protein and/or a molecule of VirD2 (preferentially covalently attached to the 5′-end of this DNA). The Vir-proteins may be present to protect the DNA inside the plant cell, take care of proper targeting towards the nucleus and will stimulate proper integration into plant DNA. Tissue will be stimulated to regenerate by the introduction of certain bioactive molecules. These bioactive molecules act as the second component. The second component is either nucleic acid, being RNA, or naked DNA with a small chance of becoming integrated in the genome, or (modified) protein product.

The nucleic acids or proteins (second component) may be introduced mixed with the first component by the methods known in art, like particle gun bombardment, electroporation, micro-injection or other techniques described in the introduction. Both components have to be present in the plant cell together in sufficient quantities, but the ratio between the two components may vary depending on the species and the preferred number of integration's of the trait in the plant DNA. The second component will preferably be lost during the regeneration process and is therefore only transiently present, whereas the first component has a high change of becoming integrated into the plant genome. The second component is a nucleic acid or a mixture of nucleic acids that will produce proteins that stimulate the regeneration process and reduce or eliminate the use of exogenously added planthormones or is the protein product or a mixture of products of these nucleic acids or their modified forms or a mixture of both. Examples of molecules with the above described characteristics are proteins, or genes coding for proteins involved in the regulation of plant development or perception of plant hormones. The main advantages of the this t/r-system are, as explained with the example of FIG. 20:

-   -   only the trait is introduced into the plant DNA; apart from the         T-DNA borders (Only in the case when VIR proteins are used, it         is necessary to include T-DNA borders onto the trait DNA), if         present, no other unwanted DNA, like a selection marker, is         present. In order to allow the process of homologous         recombination of the trait DNA into the corresponding endogenous         DNA on the plant genome, genes or gene products encoding At R51,         AtRAD51 or RecA or gene products with similar function can be         applied in the second component in order to result in transient         expression of the recombinase. After targeting and localized         integration of the trait DNA, the recombinase is lost.     -   the principle of regeneration is universally applicable     -   the amount of exogenous plant hormones for regeneration can be         reduced or omitted         active selection is not necessary as mainly transformed cells         will regenerate.

Said gene involved in the regulation of plant development can be selected from a great many genes already known, or yet to be determined, to be involved in regeneration. Examples of such genes are clavata (Clark et al., 1997, Cell 89, 575-585) and primordia timing genes (Mordhorst et al, 1998 Genetics 149, 549-563), which are stimulating regeneration when inactivated, Leafy-Cotelydon gene (LEC, Lotan et al., 1998, Cell 93, 1195-1205), the KAPP gene (Stone et al., 1994, Science 266, 793-795; Stone et al., 1998, Plant Physiol. 117, 1217-1225), IPT (Morris, R. O., 1986 Annu. Rev. Plant Physiol. 37, 509-538), WUSCHEL (Mayer et al. 1998 Cell 95, 805-815; Schoof et al. 2000 Cell 100, 635-644), KNAT1&2 (the Arabidopsis kn1-like gene) (Chuck et al. 1996. Plant Cell 8, 1277-1289; Lincoln et al. 1994 The Plant Cell 6, 1859-1876), SHOOT MERISTEMLESS gene (Endrizzi et al. 1996 Plant J. 10, 967-979), CUP-SHAPED COTYLEDON (Aida et al. 1999 Development 126, 1563-1570), CYCLIN D (Cockcroft et al. 2000 Nature 405, 575-579; Riou-Khamlichi et al. 1999 Science 283, 1541-1544), CKI1 (Kakimoto 1996 Science 274, 982-985), AINTEGUMENTA (Mizukami and Fischer 2000 PNAS 97, 942-947; Krizek 1999 Dev. Genetics 25, 224-236), SBP-box proteins (Cardon et al. 1999 Gene 237, 91-104), CDC2a (Hemerly et al. 1993 The Plant Cell 5, 1711-1723), which are genes that stimulate regeneration when induced or overexpressed, or antagonists thereof or others that are involved in the regulation of plant development in the broadest sense, such as can be found by studying plant embryogenesis or organogenesis on the molecular level. In particular, a population of gene products involved in regeneration is represented by the intracellular signal transduction factors that are directly phosphorylated by RKS protein and thereby activated.

In a preferred embodiment, the invention provides a method according to the invention wherein said gene involved in the regulation of plant development encodes a leucine-rich repeat containing receptor-like kinase, such as present in plant database collections, with homology to the extracellular domain of the Arabidopsis RKS protein family, such as:

GB:AW011134 AW011134 ST17B03 Pinus taeda GB:LELRPGENE X95269 L. esculentum GB:AI775448 AI775448 EST256548 Lycopersicon esculentum GB:AI496325 AI496325 sb05c09.y1 Gm-c1004 Glycine GB:AI487272 AI487272 EST245594 Lycopersicon esculentum GB:AI441759 AI441759 sa82d08.y1 Gm-c1004 Glycine max GB:AI782010 AI782010 EST262889 Lycopersicon esculentum GB:AI772079 AI772079 EST253179 Lycopersicon esculentum GB:SBU62279 U62279 Sorghum bicolor GB:C22645 C22645 C22645 Oryza sativa GB:D49016 D49016 RICS15625A Oryza sativa GB:AI776399 AI776399 EST257499 Lycopersicon esculentum GB:AI776208 AI776208 EST257308 Lycopersicon esculentum GB:AI352795 AI352795 MB61-10D PZ204.BNlib Brassica napus GB:AQ578072 AQ578072 nbxb0092C18f Oryza sativa GB:C95313 C95313 C95313 Citrus unshiu Miyagawa GB:AI162893 AI162893 A026P38U Hybrid aspen GB:AI782076 AI782076 EST262955 Lycopersicon esculentum

GB:AI726177 AI726177 BNLGHi5165 Cotton

GB:AI777982 AI777982 EST258861 Lycopersicon esculentum GB:AI774881 AI774881 EST255981 Lycopersicon esculentum GB:AI896737 AI896737 EST266180 Lycopersicon esculentum GB:AI676939 AI676939 605047A07.x1 Zea mays GB:D40598 D40598 RICS2674A Oryza sativa GB:OSU82168 U82168 Oryza sativa GB:SBRLK1 Y14600 Sorghum bicolor GB:AI495359 AI495359 sa97a09.y1 Gm-c1004 Glycine max GB:C96041 C96041 C96041 Marchantia polymorpha, or such as present in plant database collections, with homology to the intracellular domain of the Arabidopsis RKS protein family, such as: GB:AI896277 AI896277 EST265720 Lycopersicon esculentum GB:AU056335 AU056335 AU056335 Oryza sativa GB:AA738546 AA738546 SbRLK4 Sorghum bicolor GB:AA738544 AA738544 SbRLK2 Sorghum bicolor GB:AA738645 AA738545 SbRLK3 Sorghum bicolor GB:SBRLK1 Y14600 Sorghum bicolor GB:AI7290900 AI729090 Gossypium hirsutum GB:AI920205 AI920205 Pinus taeda GB:AI896183. AI896183 EST265626 Lycopersicon esculentum GB:AI967314 AI967314 Lotus japonicus GB:AI730535 AI130535 BNLGHi7007 Gossypium hirsutum GB:AF078082 AF078082 Phaseolus vulgaris GB:CRPK1 Z73295 C. roseus GB:C22536 C22536 C22536 Oryza sativa GB:C22530 C22530 C22530 Oryza sativa GB:CMA010166 AJ010166 Zea mays mRNA GB:AQ271213 AQ271213 Oryza sativa, or known from Schmidt et al (1997, Development 124, 2049-2062, WO 97/43427), where for example stable transformation, but not regeneration, nor transient expression nor use in selection, of plants with SERK (RKS0) is considered. Also applicable in a method according to the invention are bacterial genes or fragments thereof such as the AK-6b gene (Wabiko et al, Plant Physiol. 1996, 939-951) or the rolABC genes (Jasik J, Plaint Science, 1997, 57-68), however, where only regeneration by stable transformation is intended, plant genes such as those disclosed herein are preferred.

In a preferred embodiment, the invention provides a method according to the invention wherein said gene involved in the regulation of plant development encodes a leucine-rich repeat containing receptor-like kinase, wherein said receptor-like kinase is a representative of a plant receptor kinase family RKS such as shown in FIG. 3.

In particular, the invention provides a method wherein said gene product or functional fragment thereof is derived from a receptor-like kinase that comprises an N-terminal signal sequence, an extracellular region comprising a leucine zipper domain, a disulphate bridge domain, a leucine rich repeat domain comprising 3-5 leucine rich repeats, a transmembrane domain, an intracellular region comprising an anchor domain, a serine/threonine kinase domain and/or a C-terminal leucine rich repeat domain.

These genes encode membrane spanning proteins having a particular function in signal transduction, thereby being prime candidate genes to provide gene products or functional fragments thereof to be employed in a method of the current invention.

In particular, the invention provides a method wherein said receptor-like kinase is encoded by a nucleic acid which in Arabidopsis thaliana comprises a sequence as shown in anyone of FIG. 4 or 8 to 20. Suitable receptor kinase-like genes from plants other than Arabidopsis thaliana, such as Daucus carota, Rosa, Gerbera, Chrysanthemum, Alstroumeria, Lilium, Tulipa, Dyanthus, Cymbidium, Gypsopays, Ficus, Calangoe, Begonia, Phalasnopsis, Rhonondendrum, Spatiphilus, Cucubitaceae, Solanaceae, and grasses such as cereals are easily found using the Arabidopsis thaliana sequences provided herein by methods known in the art. In general for each RKS gene identified in Arabidopsis thaliana a corresponding RKS gene is present in individual species of both monocotyledon as well as in dicotyledon plants. The invention provides a method wherein said receptor-like kinase is encoded by a plant derived nucleic acid corresponding or homologous to a nucleic acid which in Arabidopsis thaliana comprises a sequence as shown in anyone of FIG. 4 or 8 to 20. Corresponding or homologous RKS genes and gene products in plant species other than Arabidopsis thaliana are isolated by various approaches. For example by screening of cDNA and genomic libraries using Arabidopsis RKS cDNA probes under low stringency hybridisation/washing conditions as described above, alternatively by the use of degenerated RKS primers (for example primer combination RKS B forward and RKS E reverse as shown herein in order to amplify an exon fragment of the desired gene. Full length cDNA clones can further be obtained by race and tail PCR approaches. Also, the generation of antibodies recognising conserved or distinct and specific regions within different members of RKS gene family within a plant species allow the desired isolation. Alternatively, specific antibodies are generated that recognise one specific RKS gene product in a variety of plant species. These antibodies are used to screen cDNA expression libraries of plant species. Furthermore, it is possible to screen for RKS-homologous sequences in electronic databases. Searches are performed both on nucleotide and on amino acid level. Additionally, RKS genes and gene products in plant species other than Arabidopsis thaliana are isolated for example by two or three hybrid screenings in yeast with RKS clones in other to isolate (hetero) dimerizing members of this RKS family in similar or unrelated plant species.

In one embodiment, the invention provides a method for propagation of a plant from plant starting material wherein during regeneration of said starting material at least one signal transduction pathway for root or shoot initiation is stimulated by a recombinant gene product or functional fragment thereof derived from a gene involved in the regulation of plant development allowing reducing or omitting exogenous phytohormone addition to said culture, wherein said gene product or functional fragment thereof is introduced in at least a part of the starting material by transformation. The invention also provides the introduction of regenerating gene constructs into cells which can lead to the regeneration of the cell itself or to the induction of regeneration processes in neighbouring cells, even somatic embryos resulting from said induced cells are provided herewith. Individual transformed cells are generated that are essential for the differentiation state of surrounding cells. Introduction of such an inducing regenerator as provided herewith into plant cells results in the formation of a proliferation of neighbouring cells and the formation of new plants or parts thereof from these proliferating cell masses. The originally transformed plant is not necessarily included in the proliferation process itself an is therefore not necessarily part in the resulting regenerating plants or parts thereof. This specific from of induced regeneration of neighbouring cells provide herewith gives the option to regenerate plants that do not contain the introduced gene or gene product, and therefore represents a method to induce regeneration without the necessity to introduce gene products into an originating cell population and having to maintain these gene products or nucleic acids encoding therefore. An example of the process of induced induction is shown in FIG. 6F, where a single GUS positive cell marks the original introduction site for the bombarded DNA constructs. Above this cell, a proliferating cell mass has been formed that is clearly GUS negative. On top of this induced proliferated cell mass, we could detect several structures that morphologically represent somatic embryos. These somatic embryos develop from the borders of the proliferating cell mass as previously described (Schmidt et al. 1997, Development 124, 12049-2062). Somatic embryos provide an excellent source of regenerating plant since all the organs and plant parts are formed by similar processes as take place during zygotic embryogenesis. This observation clearly indicates the potential of this class of regenerating molecules to induce a proliferating, non-transformed cell mass from which new plantlets can be regenerated. It provides the means to induce somatic embryos directly on living plant tissues, even without the prior need to introduce an in vitro culture procedure.

Again, transformation as provided here can be thus either in a stable fashion where the introduced genetic information or nucleic acid is integrated into the nuclear, chloroplast or mitochondrial genome, and is either constitutively or inducibly expressed but preferably is transient, wherein the nucleic acid is not introduced into the genome and gets lost after a certain period after introduction. Transformation of recombinant DNA or RNA into the cell or protoplast can take place in various ways using protocols known in the art, such as by particle bombardment, micro-injection, Agrobacterium-mediated transformation, viral-mediated transformation, bacterial conjugation, electroporation, osmotic shock, vesicle transport or by direct gene transfer, with or without the addition of a proteinaceous substance bound to the nucleic acid molecule. Integration of a proteinaceous substance into cells or protoplast can be facilitated along the lines of the transformation protocols as described above. A cell or protoplast thus having been provided with a gene product (i.e. a DNA, RNA or proteinaceous substance or functional fragment thereof) derived from a gene involved in the regulation of plant development can now regenerate on its own, allowing reducing or omitting exogenous phytohormone addition to the culture that comprises that cell or protoplast. The process of vegetative propagation is hereby very much simplified, large numbers of plants with an identical genetic background can now be obtained staring from starting material with the desired characteristics.

In a preferred embodiment, the present invention provides a method for propagation of a plant from plant starting material wherein said starting material comprises a cell or protoplast transformed with a desired nucleic acid sequence intended to provide the resulting transgenic plant arising from that cell or protoplast with desirable characteristics. Such a cell or protoplast, according to the invention having been provided with a gene product (i.e. a DNA, RNA or proteinaceous substance or functional fragment thereof), for example derived from a gene involved in the regulation of plant development can now regenerate on its own, allowing reducing or omitting exogenous phytohormone addition to the culture that comprises that transformed cell or protoplast. Selection for regenerating cells or tissues after the transformation of the desired sequence together with the regenerating gene product results in the recovery of only those plants or plant material that contain the desired nucleic acid sequence, preferably integrated in a stable fashion in the plant's genome, and the regenerating gene product, thereby providing a selection of the desired transgenic plant based on the selective regeneration of the transformed starting material.

In a preferred embodiment, the invention provides a method wherein the regenerating gene product is only transiently expressed, wherein the regenerating gene product or its coding sequence is not introduced into the genome and gets lost after a certain period after introduction, hereby providing an essentially marker-free transgenic plant as end-product, containing only the desired transgenic nucleic acid, and not the nucleic acid encoding the selection marker used: the regenerating gene product.

Furthermore, the invention provides plant or plant material obtainable by a method according to the invention, propagated along the lines or using a method herein disclosed. In particular, the invention provides a plant or plant material obtainable by in vitro vegetative or seedless propagation according to the invention from plant starting material, for example using single-node cuttings, axillary branching, regeneration of adventitious organs (roots or shoots), or starting material such as explants or callus tissue or suspensions of, or even single, cells or protoplasts, in particular wherein said starting material comprises transgenic material, said transgenic plant or plant material according to the invention preferably being free of a selection marker gene.

The invention furthermore provides an isolated and/or recombinant nucleic acid encoding a receptor-like kinase or a functional fragment or functional equivalent thereof, corresponding to or capable of hybridising to a nucleic acid molecule as shown in anyone of FIG. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or its complementary nucleic acid. Such a nucleic is obtained as described above. In a preferred embodiment, such a nucleic acid is at least 75% homologous, preferably at least 85%, more preferably at least 90%, or most preferably at least 95% homologous to a nucleic acid molecule or to a functional equivalent or functional fragment thereof, as shown in anyone of FIG. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or its complementary nucleic acid, for example derived from Arabidopsis thaliana.

Also, the invention provides a vector comprising a nucleic acid according to the invention. Such a vector is preferably capably of providing stably or transient transformation of a cell by providing said cell with nucleic acid (DNA or RNA) or protein derived from a nucleic acid according to the invention. A variety of methods to provide cells with nucleic acid or protein are known, such as electroporation, liposome-mediated transfer, micro-injection, particle gun bombardment or bacteria-mediated transfer. RNA can for example be produced in vitro from appropriate vector constructs incorporating sites such as SP6, T7 or T3. Protein is produced in vitro in for example yeast or bacterial or insect cells, or other appropriate cells known in the art. DNA can be delivered as linear or circular DNA, possibly placed in a suitable vector for propagation.

1. Furthermore, the invention provides a host cell comprising a nucleic acid or a vector according to the invention. In a preferred embodiment, such a host cell is a transformed cell additionally comprising a desired, but most times totally unrelated, nucleic acid sequence, preferably integrated in a stable fashion in its genome. Even more preferred is a host cell according to the invention wherein the nucleic acid or vector according to the invention is only transiently expressed. Of course it is preferred to use a nucleic acid, vector or host cell according to the invention for use in a culture method as provided by the invention. The invention also provides a method for determining a developmental stage of a plant comprising detecting in said plant or parts thereof a nucleic acid or a proteinaceous substance according to the invention. Said detection is thus aimed at using receptor kinase genes or gene products belonging to the RKS family, or fragments thereof, as markers for plant development.

The invention furthermore provides an isolated or recombinant proteinaceous substance comprising an amino acid sequence as shown in anyone of FIG. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or a functional equivalent or functional fragment thereof. Proteinaceous substance herein is defined as a substance comprising a peptide, polypeptide or protein, optionally having been modified by for example glycosylation, myristilation, phosphorylation, the addition of lipids, by homologous or heterologous di- or multimerisation, or any other (posttranslational) modifications known in the art.

Based on sequence composition, the N-terminal domain of predicted amino acid sequences of the RKS gene family represents a signal peptide, indicating that this region of the protein is extracellular. The length of this signal sequence and the predicted cleavage sites have been established using a prediction program: http://genome.cbs.dtu.dk/services/SignalP/. This domain is followed by a short domain containing a number of leucine residues, seperated from each other by 7 amino acid residues. Based on the conservation of these leucines in an amphipathic helix, this domain represents a leucine zipper domain that mediates protein dimerization through formation of a short coiled-coil structure (Landschultz W H, Johnson P F, and McKnight sSL (1988) Science 240, 1759-1764). In RKS proteins, this leucine zipper domain is likely to be involved in receptor hetero/homo dimerization. The next domain contains 2 conserved cysteine residues that forms a disulphate bridge. The subsequent domain represents a leucine rich repeat (LRR) region with 3-5 LRRs of approximately 24 amino acids each. In animals, this domain is known to be involved in protein-protein interactions (Kobe B and Deisenhofer J (1994) TIBS 19, 415-420). In plants the extracellular LRR region is predicted to be necessary for ligand and elicitor binding. At the C-terminal part of the LRR region of most RKS proteins, another conserved couple of cysteine residues is involved in the formation of another disulphate bridge. At both ends, the LRR domain is thus surrounded by two disulphate bridges. The next domain contains a relatively high number of P and S amino acid residues, and shows similarity with cell wall proteins like extensins. Prediction server programs like http://genome.cbs.dtu.dk/services/NetOGlyc/ indicate the presence of multiple O-glycosylation sites within this domain. This domain might have similar functions as extensins and provide interaction sites with multiple cell wall components, thus forming a stable immobilised interaction with the cell wall in which the complete extracellular region of RKS proteins is embedded. The next domain represents a single transmembrane helical domain, as predicted by the program http://genome.cbs.dtu.dk/services/TMHMM-1.0/. The end of this domain, and the beginning of the intracellular cytoplasmic domain, contains a small number of basic K and R residues. The next domain is relatively acidic. The next large domain shows extensive homology with the family of plant serine, threonine receptor kinases. Autophosphorylation studies on SERK (Schmidt et al. 1997) have shown that this domain shows serine, threonine kinase activity. Within the kinase domain, several RKS proteins like RKS0 and RKS8 contain a putative 14-3-3 binding site represented by the core sequence RxpSxP, in which x represents any amino acid (Yaffe M B, Rittinger K, Volinia S, Caron P R, Aitken A, Leffers H, Gamblin S J, Smerdon S J and Cantley L C (1997) Cell 91, 961-971). (Auto)phosphorylation of the S residue within this sequence as a result of ligand-mediated receptor-kinase activation would thus allow the binding and subsequent activation of 14-3-3 proteins. The next domain has an unknown function although the conservation of WD pair residues suggests a function of a docking site for other proteins. The C-terminal intracellular domain contains again part of a single LRR sequence, and might therefore be involved in protein-protein interactions. Preferably such a proteinaceous substance according to the invention is encoded by a nucleic acid according to the invention or produced by a host cell according to the invention.

In particular, the invention provides a proteinaceous substance for use in a culture method according to the invention. Introduction of a proteinaceous substance into cells or protoplast can be facilitated along the lines of the transformation protocols as known in the art. A variety of methods are known, such as micro-injection, particle gun bombardment or bacteria-mediated transfer. A cell or protoplast thus having been provided with a proteinaceous substance or functional fragment thereof derived from a gene involved in the regulation of plant development can now regenerate on its own, allowing reducing or omitting exogenous phytohormone addition to the culture that comprises that cell or protoplast. The process of vegetative propagation is hereby very much simplified, large numbers of plants with an identical genetic background can now be obtained staring from starting material with the desired characteristics. Proteins or peptides, encoded for by the RKS genes, are produced by expressing the corresponding cDNA sequences, or parts thereof in vitro or in an in vivo expression system in E. coli yeast, Baculovirus or animal cell cultures. The expressed protein sequences are purified using affinity column purification using recombinant Tag sequences attached to the proteins like (HIS)6 tags. Tags are removed after purification by proteolytic cleavage. The resulting protein sequence encodes a functionally active receptor-kinase, or a derivative thereof. In a preferred embodiment, the protein contains a (constitutive) active kinase domain. The purified recombinant protein is introduced into plant cells in order to induce regeneration from these cells in a transient fashion. Proteins are introduced by methods similar as described for the introduction of nucleotide sequences, such as liposome-mediated transfer, micro-injection, electroporation, particle gun bombardment or bacteria-mediated transfer. If so desired, modification of recombinant proteins like glycosylation, disulphate bridge formation, phosphorylation etc. can be optimized in order to obtain an optimal efficiency in protein stability and activity.

Also, the invention provides an isolated or synthetic antibody specifically recognising a proteinaceous substance according to the invention. Such an antibody is for example obtainable by immunising an experimental animal with a proteinaceous substance according to the invention or an immunogenic fragment or equivalent thereof and harvesting polyclonal antibodies from said immunised animal, or obtainable by other methods known in the art such as by producing monoclonal antibodies, or (single chain) antibodies or binding proteins expressed from recombinant nucleic acid derived from a nucleic acid library, for example obtainable via phage display techniques. Such an antibody can advantageously be used in a culture method according to the invention, for example to identify cells comprising a regenerating gene product as identified above. With such an antibody, the invention also provides a proteinaceous substance specifically recognisable by such an antibody according to the invention, for example obtainable via immunoprecipitation, Western Blotting, or other immunological techniques known in the art. Also, the generation of such antibodies recognising conserved or distinct and specific regions within different members of RKS gene family within a plant species allow the desired isolation of RKS-homologues or recognise a specific RKS gene product in a variety of plant species. These antibodies are also used to screen cDNA expression libraries of plant species to screen for RKS-homologues. The invention, and use as provided of a nucleic acid, a vector, a host cell, a proteinaceous substance or an antibody according to the invention in a method according to the invention is further explained in the detailed description without limiting the invention.

DETAILED DESCRIPTION

In order to isolate genes involved in the developmental regulation of regeneration in plants, the different members of a family of genes were identified of which the expression was present in developing influorescenses. Within this tissue a large number of different organ primordia are initiated from the influorescence meristems. As a model plant species Arabidopsis thaliana was chosen, based on the presence of many well characterized genetic mutations and the availability of genetic information in databases.

The differentiation stage is highly stable in vivo, yet in response to nuclear transplantation or cell fusion, the nuclei of differentiated cells exhibit a remarkable capacity to change, both in animal and in plant cells (Blau, 1989). The ability to change the differentiation stage provides cells and tissues with the ability to adapt towards their environment. Normally only a small number of stem cells have the ability to differentiate into different cell types. In plants, the only cells that are truly totipotent are the zygotes, consisting of fused egg cells and sperm. From these dipoid totipotent cells all other differentiated cell types are derived.

Regeneration is a vegetative reproduction or repair strategy observed in a large number of animal and plant species. Regeneration in plants is defined as the formation of new tissues containing both root and shoot meristems, separate shoot or root meristems, plant organs or organ primordia from individual cells or groups of cells. Regeneration mimics the process of normal cellular and organ differentiation that takes place during plant development and results in the formation of the different plant organs. However, plant cells or groups of cells that under normal conditions are unable to initiate the formation of certain plant organs, meristems or organ primordia can be stimulated by either extracellular stimuli or intracellular modification of the differentiation stage of the cell. Regeneration can take place under either in vivo or in vitro conditions. Regeneration does not include the process of apomixis, wherein specific forms of vegetative plant reproduction are taking place in seeds. Extracellular diffusible factors have shown to be essential for cellular redifferentiation in plant cells (Siegel and Verbeke, 1989). The perception of these signals at the cellular surface and the intracellular signal transduction that finally result in changes in transcriptional regulation provides cells with the ability to respond to such extracellular stimuli.

In a search for gene products with the ability to regulate cellular differentiation we concentrated on genes involved in perception and transmission of intercellular differentiation signalling. Extracellular signals in animal cells are normally perceived by an high affinity binding compound, the sensor molecule. Extracellular signalling factors are further referred to as ligands and their cellular binding partners are defined as receptors. Upon binding, the extracellular signal can result in modification of the receptor, resulting in transmission of the signal over the cellular membrane. Cell surface receptors contain an extracellular ligand binding domain, a transmembrane domain and an intracellular domain involved in transmission of signals to the intracellular signal transduction components (Walker, 1994). SERK represents a member of the large group of transmembrane receptor kinases with various functions in plants and animals. Many of these gene products are known to be involved in cellular differentiation processes like Clavata 1 (Clark et al. 1997) or Erecta (Torii et al. 1996). Overexpression or mutation of these genes in plants result in morphological changes in plant organs or plant cells.

The Somatic Embryogenesis Receptor-like Kinase SERK was originally identified as a marker for embryogenic cells, both in vivo, and in vitro. (Schmidt et al. 1997a). Expression of the SERK gene was correlated with the ability to form somatic embryos, a process in which plants are formed from somatic cells through the same morphological, cytological and molecular sequence of stages of embryogenesis as zygotic embryos.

Transmembrane proteins like receptor kinases provide a set of candidate key regulator gene products that are involved in organ or cellular differentiation. In a search for gene products with the ability to modulate the differentiated we searched for receptor-kinase genes expressed in a plant tissues with a large variety of cellular differentiation processes, the influorescense meristem. In a screen for gene products involved in the regulation of the differentiation stage of cells we identified a complete family of receptor-like kinases.

Identification of a new family of receptor-like kinases in Arabidopsis thaliana, the RKS gene family.

In genomic databases of Arabidopsis (accession http://genome-www2.stanford.edu/cgi-bin/AtDB/nph-blast2atdb), a small number of sequences was identified with homology to the Arabidopsis SERK sequence (Schmidt et al. 1997b). These sequences showed homology on nucleotide and predicted amino acid level and were further defined as Receptor Kinases-like SERK (RKS) genes. The initially identified sequences are further defined as RKS₁₋₅. Based on these five RKS sequences a set of degenerated DNA primers was designed that allowed amplification of possible RKS gene fragments from Arabidopsis.

Primer RKS B forward: 5′-CC[C/G] AAG AT[C/T] AT[A/T] CAC CG[A/C/T] GAT GT[A/C/G] AA[A/G] GC-3′ Primer RKS E reverse 5′-CC[A/G] [A/T]A[A/C/G/T] CC[A/G] AA[A/G] ACA TCG GTT TTC TC-3′

These sequences are based on conserved parts within the nucleotides encoding one exon of the kinase domain. PCR amplification reactions (60 sec. 94° C.; 60 sec. 50° C.; 90 sec. 72° C.)×40 cycli. were performed with 100 ng of genomic DNA as a template. The resulting PCR products consisted of 209 bp DNA fragments. After cloning in a pGEM-T (Promega) vector, a total of 21 different clones was analysed in order to identify the amplified nucleotide sequences. Removal of the degenerated primer sequences resulted in sequences of 154 nucleotides. Apart from the sequences of RKS1-4 and the SERK gene, a total of 4 new unidentified RKS homologous sequences were identified, further defined as RKS6-10. Sequences from the RKS5 gene were not identified in this screen.

Number of clones isolated and sequenced for different RKS genes followed by time(s) identified in genomic PCR.

RKS1 1 RKS2 4 RKS3 2 RKS4 5 RKS5 0 RKS6 2 RKS7 1 RKS8 2 RKS103 SERK/RKS0 1

These results indicated the presence of at least 9 different sequences with homology to the conserved kinase domain of the predicted RKS genes (apart from SERK) on the Arabidopsis genome (FIG. 1). In order to confirm these data, the fragment of one of the isolated RKS genes was used as a probe in a Southern blot (FIG. 2). Low stringency hybridization confirmed the presence of a number of sequences related to the probe fragment. Under the stringency used (see Materials and Methods) a total of approximately 5 hybridizing bands could be observed, indicating the presence of a small RKS gene family in Arabidopsis.

RKS gene expression in Arabidopsis inflorescence tissues.

In order to test whether RKS genes are expressed in tissues where formation of primordia and organs is initiated, RT-PCR reactions were performed on inflorescences. The same combination of PCR primers for RKS fragment amplification was used as described for the genomic PCR reactions. Due to the absence of intron sequences in the described nucleotide fragments, the resulting product was again 209 bp. Starting from the first strand cDNA, a standard PCR reaction was performed for (60 sec. 94° C.; 60 sec. 50° C.; 90 sec. 72° C.)×40 cycli. In order to obtain a sufficient large amounts of amplified product, a reamplification was performed under similar conditions, using 10% of the mix from the first RT-PCR amplification reaction mix as a template. After cloning in a pGEM-T vector, a total of 21 different clones was sequenced in order to identify the amplified sequences. Removal of the degenerated primer sequences resulted in sequences of 154 nucleotides (FIG. 1).

Number of RT-PCR clones isolated and sequenced for different RKS genes followed by time(s) RT-PCR product identified from influorescence tissue:

RKS1 0 RKS2 0 RKS3 2 RKS4 5 RKS5 0 RKS6 0 RKS7 1 RKS8 2 RKS104 RKS112 RKS123 RKS131 RKS141 SERK/RKS0 0 RKS 14

These results indicated the presence of at least 14 different sequences with homology to the conserved kinase domain of the predicted RKS genes (apart from SERK) on the Arabidopsis genome (FIG. 1). Within influorescenses, at least 9 RKS-like genes were expressed. Within this experiment, expression of is RKS 0, 1, 2, 5 and 6 in inflorescences could not be confirmed. Homology between the different RKS sequences was performed using ALLIGMENT software from Geneworks 2.2 (FIG. 3). At least three different subgroups could be visualized of the RKS gene family, representing RKS 2 and RKS6 in subgroup 1, RKS 4, 11, 1, 5, 14 and 7 in subgroup 2 and RKS 0, 8, 10, 12 and 13 in subgroup 3. These results confirmed the hybridization patterns, observed with genomic Southerns hybridized with a member of the RKS subgroup 3 (FIG. 2). A total of 5 hybridizing bands could be observed, that were likely to represent the genes from RKS 0, 8, 10, 12 and 13.

In order to investigate whether the isolated PCR fragments represented parts of complete RKS genes, full length and partial cDNA clones homologous to these PCR fragments were isolated and characterized.

Isolation and Characterization of the RKS Gene Products in Arabidopsis

A cDNA library from Arabidopsis thaliana Colombia wild type was used to isolate cDNA clones hybridizing with the PCR amplified RKS gene fragments. The consisted of a BRL λZipLox vector containing SalI, NotI linked cDNA inserts from different plant organs (including siliques, flowers, stems, rosette leaves and roots.

Filter hybridization, purification of plaques hybridizing under stringent conditions (65° C., 0.1SSC) with the different RKS fragment probes and finally nucleotide sequence analysis resulted in the characterization of a number of RKS cDNA clones. The predicted amino acid sequences of these clones confirmed that the gene products represent members of the RKS plant receptor kinase family RKS. The sequences from the clones identified by the cDNA library were compared and combined with sequence information from the database http://arabidopsis.org/blast/. Apart from 14 different full length cDNA clones a number of 4 different partial clones were identified.

Overexpression of RKS Gene Products in Transgenic Arabidopsis

Transformation of plasmid DNA into plant cells was performed using A. tumefaciens C58C1. The binary vector used consisted of pGREEN, pGREEN1K or RKS expression constructs. Bacterial colonies were grown on LB agar plates containing 20 mg/L gentamycin, 50 mg/L kanamycin and 50 mg/L rifampicin. Five colonies were used to inoculate 50 ml of LB medium containing 50 mg/L kanamycin and 50 mg/L rifampicin. After 16 hours of incubation at 30° C. cells were concentrated by centrifugation and resuspended in 10 ml infiltration medium (consisting of 5% sucrose and 0.05% Silwett L-77 in water. A helper plasmid, necessary for transformation, consisted of the vector pJIC Sa-Rep and was co-transformed together with the pGREEN vector. After electroporation and incubation for 2 hours at 30° C., cells were plated onto LB plates with 50 mg/L rifampicin en 50 mg/L kanamycin. Arabidopsis thaliana wild-type WS cultivar was transformed following the floral dip protocol (Clough and Bent, 1998). In short, the influorescences of young Arabidopsis WS plants grown under long day conditions (16 hours light, 8 hours dark) were dipped for 10 seconds in 10 ml of infiltration solution. Plants were grown further under long day conditions and seeds were harvested after an additional 3-5 weeks. Seeds were surface sterilized in 4% bleach solution for 15 minutes and after extensive washing in sterile water, plated on ½MS plates with 60 mg/L kanamycin. After 10 days incubation under long day conditions, transgenic kanamycin resistant seedlings were isolated and planted on soil for further non-sterile growth under standard long day greenhouse conditions. This infiltration protocol routinely resulted in approximately 1% transformed seeds for each of the RKS gene constructs used.

Regeneration of Arabidopsis Plants After RKS Gene Transformation

Arabidopsis T2 seeds; obtained from plants infiltrated with A. tumefaciens containing empty pGREEN vectors or pGREEN1K vectors including RKS genes under the control of a 35S promoter, were surface sterilized and added to 40 ml ½MS medium culture to which 1 mg/L 2,4-D was added. After three days of stratification at 4° C., the cultures were incubated on a shaker under long day conditions in a climate room of 20° C. for 0-18 days to induce cell proliferation. At different time intervals, seedlings were isolated from the culture, washed and transferred onto ½MS agarplates without 2,4-D or any other hormones. Incubation in the climate room was continued under long day conditions for 4 more weeks. In the absence of RKS genes in the transformed binary vector, no regeneration of plantlets could be observed (FIG. 5C). However, in the presence of RKS gene expression, regenerating plants could be observed that originated from the proliferating cell mass (FIGS. 5A,B). Different RKS gene constructs showed the ability to regenerate shoot meristems and leaves. The ability to induce regeneration varied between individual integration events and between RKS gene constructs (FIG. 5A versus 5B). At this timepoint of 4 weeks of regeneration, plantlets were transferred directly to non-sterile soil and grown for another 4-6 weeks under long day conditions. Fertile, seed setting plants could be obtained from the regenerated plantlets as shown in FIGS. 5A,B.

20 μg of vector DNA for biolistic DNA delivery into Arabidopsis tissue was mixed with a ballistic suspension mix: 10 mg of gold (Aldrich Chem, Co. Gold 1.5-3 micron), 30 μl 5M NaCl, 5 μl 2M Tris pH 8, 965 μL water, 100 μl 0.1M spermidine, 100 μL 25% PEG, 100 μl 2.5M CaCl₂. The suspension was incubated at room temp for 10 min, and centrifuged. The resulting pellet was washed twice with ethanol and resuspended into 200 μl icecold 99.8% ethanol. For each microprojectile bombardment, 10 μl of the gold-coated DNA was used. Bombardment conditions for the HELIUM GUN 461 were: helium pressure 6 bar, vacuum to 50 mbar and 9 cm distance of the tissue from the filter. 0.1 mm mesh size screen was used between tissue and filter, 3 cm distance of the screen from the filter. After bombardment, the Arabidopsis plants were cultured for a period of 3 weeks under long day conditions.

Regeneration in Nicotiana tabacum Induced by Expression of Regeneration-Stimulating Gene Products

20 microgram of plasmid DNA was transferred into cells of tobacco (NTSR1) leaves, using biolistic bombardment with gold particles coated with DNA. Leaf discs were subsequently submerged in liquid MS30 medium (MS medium 30 g sucrose/l, Murashige and Skoog 1962) containing 1 mg/l kinetin and incubated on a rotary shaker (250 rpm) for 14 days. Leaves were then transferred to plates with MS30 plates, 0.8% agar. All incubations have been performed at 20° C. with 16 hours light, 8 hours dark. Control experiments with empty or control vectors never gave rise to shoot formation. Regenerating plantlets appeared as a result of particle bombardment with regenerating DNA constructs as shown in FIG. 6A-C. The transient nature of the introduced construct could be confirmed for 9 out of 10 different regenerants obtained from bombarded tissue (FIG. 6D).

Induction of Cell Proliferation in Arabidopsis thaliana Induced by Expression of Regeneration Inducing Gene Products

In order to identify the earlier stages of regeneration after particle bombardment the formation of cellular proliferation was studied as a result of the activity of the regenerating gene product. Single regenerating constructs or combinations of such DNA constructs were bombarded onto two weeks old seedlings of Arabidopsis thaliana grown on MS agar plates. Between one and three weeks thereafter the formation of multicellular structures arising from the surface of bombarded rosette leaves could be observed (FIG. 6E-H). Bombardments with empty control vectors never gave rise to the formation of these structures. Interestingly, the proliferating cell mass originating from bombardment with a GT-W-20S construct developed somatic embryos as a clear indication of regeneration by the process of somatic embryogenesis.

Somatic embryogenesis was hereby not depending on a tissue culture state of the originating tissue but could be directly initiated on adult leaves still attached to the parent plant. Combinations of different regenerating constructs coated on the same gold particle before bombardment allowed also the process of cellular proliferation to be initiated (FIG. 6G). Multiple loci of proliferated tissue could be observed on individual leaves after the different regenerating constructs (FIG. 6H), indicating that the frequency of regeneration was relatively high when using combinations of regenerating constructs in contrast to bombardments with individual regenerants.

Materials and Methods Southern Blotting

10 μg of genomic DNA from Arabidopsis thaliana wildtype was digested with different restriction enzymes. Fragment DNA was size separated on a 0.9% agarosegel. DNA purination was performed in 0.6M NaCl with 0.4M NaOH. Capillairy blotting was performed onto Hybond N+ membranes. Membranes are hybridized overnight at 65° C. in C&G hybridization mix (Church and Gilbert, 1985) and subsequently washed at 65° C. with 5SSC, 0.1% SDS. For detection of radioactivity, the Phosphorimager 425 (Molecular Dynamics) was used in combination with phosphoscreen exposure casettes and ImageQuaNT software.

DNA Fragment Purification

DE81 paper (Whatmann) was used for isolation of DNA fragments from agarose gels. Paper segments were introduced into the agarosegel just behind the desired DNA fragments (which were visualized under long wave UV with ethidium bromide staining). Electrophoresis was performed for 10 minutes at 10 V/cm gel and the DE81 paper to which the DNA was bound was recovered from the gel. Paper fragments were washed extensively in Low Salt Buffer (LSB) and subsequently DNA was removed from the paper in a small volume of High Salt Buffer (HSB).

LSB (Low Salt Buffer): HSB (High Salt Buffer):  10 mM Tris pH 7.5 10 mM Tris pH 7.5  1 mM EDTA  1 mM EDTA 100 mM LiCl2  1 M LiCl2 20% Ethanol

Radioactive Probes

Purified DNA fragments were radiolabelled with 32P-dCTP following a random primed labelling:

50 ng of fragment DNA in 27 μl water is denatured for 5 min. at 100° C. On ice, 21 μl of GAT mix was added: 0.67 M Hepes, 0.17 M Tris, 17 mM MgCl2, 33 mg/ml acetylated BSA, 25 mg/ml random hexamer primers, 33 mM b-mercapto-ethanol, 5 mM dNTP's (G+A+T) without dCTP. 2 μl dCTP and 2 μl Klenow (1 U/μl) was added, mixed and incubation was performed for 60 min. at 25° C.

Genomic PCR

Genomic DNA was isolated from wild type Arabidopsis thaliana plants using the protocol of Klimyuk et al. (1993). All PCR reactions were performed in a Thermal Cycler from Perkin Elmer.

PCR amplification reactions were performed under standard conditions using the following mix: 100 ng genomic template DNA in 5 μl water, denatured for 5 min. at 100° C. On ice the following components were added: 2 μl primer B (10 μM) en 2 ml primer E (10 μM), 1 μl dNTP's (10 mM), 5 μl 10× Taq buffer (Boehringer Mannheim), 0.1 ml Taq polymerase, 5 Units/μl (Boehringer Mannheim), 35 μl water. Paraffin oil was added to the surface in a volume of 20 μl and amplification was performed under the following conditions: (60 sec. 94° C., 60 sec. 50° C., 90 sec. 72° C.)×40 cycli. PCR products were routinely purified using the High Pure-PCR product purification kit (Boehringer Mannheim). Purified DNA was cloned in a five-fold molar excess in the pGEM-T Easy vector (Promega) following standard protocols and reaction mixes as supplied within the reaction kit.

RT-PCR

Inflorescences from Arabidopsis thaliana was used as source material to isolate total RNA following the protocol of Siebert and Chenchik (1993) 2.5 μg of total RNA in 10 μl of water was linearized by 1 min. incubation at 100° C., followed by the addition of the following components on ice:

-   -   2 μl (10 pmol) dT race primer 5′-GAC TCG AGT CGA CAT CGA TTT TTT         TTT TTT TT-3′     -   1 μl dNTP's (10 mM)     -   4 μl 5×RT buffer (Boehringer Mannheim)     -   0.8 μl reverse transcriptase M-MuLV Expand (Boehringer Mannheim)     -   2 μl 100 mM DTT

Incubation was performed for 60 min. at 42° C., diluted with an equal amount of RNAse free water and stored at −20° C. 2 μl of first strand (=125 ng) was used in PCR reactions, using the RKS degenerated primers B and E. 2 μl primer B (10 μM) en 2 μl primer E (10 μM), 1 μl dNTP's (10 mM), 5 μl 10× Taq buffer (Boehringer Mannheim), 0.1 ml Taq polymerase, 5 Units/μl (Boehringer Mannheim), 38 μl water.

Paraffin oil was added to the surface in a volume of 20 μl and amplification was performed under the following conditions: (60 sec. 94° C., 60 sec. 50° C., 90 sec. 72° C.)×40 cycli. PCR products were routinely purified using the High Pure-PCR product purification kit from Boehringer Mannheim. Purified DNA was cloned in a five-fold molar excess in the pGEM-T Easy vector (Promega) following standard protocols and reaction mixes as supplied with the reaction kit.

E-coli and A. tumefaciens Transformation

Transformation of plasmid DNA into competent bacteria was performed by electroporation (Dower et al., 1988), using a Genepulser (Biorad). Conditions for electroporation were as follows: 1.5 kV, 25 mF and 200 W in standard cuvettes. Directly after transformation, cells were incubated for 90 min. at 37° C. in SOC medium (Sambrook et al. 1989). The bacterial suspension was plated on selective agar plates and incubated overnight at 37° C. (E. coli) or for two days at 30° C. (A. tumefaciens) in order to visualize transgenic bacterial colonies.

Nucleotide Sequence Analysis

Plasmid DNA was isolated from E. coli by standard boiling method protocol (Sambrook et al. 1989) followed by a subsequent purification with the PCR product purification kit from Boehringer Mannheim. Plasmids were sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Core Kit van Perkin Elmer, using standard protocols as designed for the 480 DNA Thermal Cycler. After electrophoresis on polyacrylamide gels, the results were analysed using the 373A DNA Sequencer from Applied Biosystems. Data were analysed using the software programs Sequencer 3.0, Geneworks 2.2 and DNA-strider 1.2.

cDNA Library Screening

Plating of the cλZipLox cDNA library was performed as described by the supplier protocols (GIBCO BRL), and plaque lifting and purification as described by Sambrook et al. (1989). cDNA library screening was performed using 20 duplicate filters, each containing approximately 250.000 individual plaques. Filters were screened with different RKS DNA probes representing 209 bp amplified PCR fragment. Prior to labelling, DNA fragments were isolated from the pGEM-T vector by digestion and purified twice by DE81 purification from agarose gels. Filters were hybridized under stringent conditions (0.1SSC, 65° C.). Plaques that hybridized on both filters were isolated and used for two subsequent rounds of further purification. The resulting cDNA clones were sequenced using the T7 and SP6 primers from the primer binding regions of the multiple cloning sit of the λZipLox vector. Internal oligos were designed to sequence the complete cDNA inserts of the RKS clones. Only one cDNA clone was sequenced completely for each RKS gene product identified. An alternative approach to identify and subsequently isolate cDNA clones from RKS genes was to screen the Arabidopsis genome database for RKS homologous sequences and to amplify cDNA clones by RT-PCR approach as described above using primers specific for these RKS gene products, based on the sequence data obtained from Arabidopsis genomic databases (accession http://genome-www2.stanford.edu/cgi-bin/AtDB/nph-blast2atdb). Purified RT-PCR products were cloned in a five-fold molar excess in the pGEM-T Easy vector (Promega) following standard protocols and reaction mixes as supplied with the reaction kit.

Regenerating Gene Product Expression Constructs

The CaMV 35S promoter enhanced by duplication of the −343/−90 bp region (Kay et al, 1987) was isolated from the vector pMON999 together with the NOS terminator by NotI digestion. The resulting construct was cloned into the vector pGreen (Bean et al. 1997) and the resulting binary vector is further defined as pGreen1K. RKS cDNA clones (FIG. 2) were isolated from either the pGEM-T easy vector by EcoRI digestion or from the λZipLox vector by EcoRI/BamHI digestion. The resulting cDNA fragments were cloned into respectively EcoRI digested pGreen 1K or EcoRI/BamHI digested pGreen 1K. Nucleotide sequence analysis was performed in order to test the integrity and the orientation of the RKS cDNA in the vector pGreen1K. The resulting constructs in which the different RKS₀₋₁₄ had been ligated in the sense configuration with respect to the 35S promoter are further defined as RKS expression constructs. The other regenerating gene products as previously mentioned have been cloned in a similar fashion into the pGreen expression construct under the control of a 35S promoter

Regeneration Induced by Transient Expression of RKS Gene Products

Rosette leaves and shoot meristems from 3-weeks old Arabdopsis plants grown under long day conditions were surface sterilized in a 1% bleach solution for 20 min, washed extensively with sterile water and placed on ½ MS plates solidified with 0.8% agar.

Particle Bombardment

20 μg of vector DNA for biolistic DNA delivery into plant tissue was mixed with a ballistic suspension mix: 10 mg of gold (Aldrich Chem, Co. Gold 1.5-3 micron), 30 μl 5M NaCl, 5 μl 2M Tris pH 8.0, 965 μl water, 100 μl 0.1M spermidine, 100 g 25% PEG, 100 μl 2.5M CaCl₂. The suspension was incubated at room temp. for 10 min. and centrifuged. The resulting pellet was washed twice with ethanol and resuspended into 200 μl icecold 99.8% ethanol. For each microprojectile bombardment, 10 μl of the gold-coated DNA was used. Bombardment conditions for the HELIUM GUN 461 were: helium pressure 6 bar, vacuum to 50 mbar and 9 cm distance of the tissue from the filter. 0.1 mm mesh size screen was used between tissue and filter, 3 cm distance of the screen from the filter.

REFERENCES

-   Bean S J, Gooding P S, Mullineaux P M and Davies D R (1997) Plant     Cell Reports 16, 513-519. -   Blau H M (1989) Trends in Genetics 5, 268-272. -   Church C and Gilbert K (1985) Proc. Natl. Acad. Sci. USA 81,     1991-1995. -   Clark S E, Williams R W and Meyerowitz (1997) Cell 89, 575-585. -   Dower W J et al. (1988) Nucl. Acid Res. 16, 6127-6145. -   Kay et al. (1987) Science 236, 1299-1302. -   Klimyuk V I, Carroll B J, Thomas C M and Jones J D G (1993) Plant J.     3, 493-494. -   Sambrook, Fritsch E F and Maniatis T. (1989) Molecular cloning: a     laboratory manual, Cold Spring Harbor Laboratory, New York. -   Schmidt E D L, Hecht V, van Holst G J, de Vries S C (1997b)     production of apomictic seed. International publication number     WO97/43427. -   Schmidt E D L, Guzzo F, Toonen M, de Vries S C (1997a) Development     124, 2049-2062. -   Siebert P D and Chenchik A (1993) Nucl. Acid Res. 21, 2019-2020. -   Siegel B A and Verbeke J A 1989, Science 244, 580-582. -   Torii K U, Mitsukawa N, Oosumi T, Matsuura Y, Yokoyama R, Whittier R     F and Komeda Y (1996) Plant Cell 8, 735-746. -   Walker J C (1994) Plant Molecular Biology 26, 1599-1609. -   Murashige T. and Skoog F. (1962). A revised medium for rapid growth     and bioassays with tobacco tissue cultures. Physiol. Plant. 15,     473-496

FIGURE LEGENDS

FIG. 1 depicts the different 154 bp PCR fragments as amplified with the degenerated forward and reverse RKS primers B and E, as shown in Material and Methods. The sequence of the RKS0 fragment is identical with the corresponding region of the Arabidopsis SERK gene. The nucleotide sequences representing the primer sequences have been deleted from the original 209 bp PCR products in this figure.

FIG. 2.

Genomic Southern blot of Arabidopsis thaliana genomic DNA digested with different restriction enzymes. 10 μg of genomic digested DNA is loaded in each lane. Low stringency hybridization (65° C., 5SSC) is performed with a 209 bp PCR fragment encoding part of the kinase domain of RKS0.

FIG. 3.

Homologies between the 154 bp fragments as amplified from Arabidopsis with the degenerated RKS primers B and E, shown in FIG. 1. At least three different subgroups can be visualized of the RKS gene family, representing RKS 2 and RKS6 in subgroup 1, RKS 4, 11, 1, 5, 14 and 7 in subgroup 2 and RKS 0, 8, 10, 12 and 13 in subgroup 3. Alignments were performed using DNA Strider 1.2 software.

FIG. 4A

Arabidopsis thaliana RKS0 cDNA

The start codon has been indicated by bold capitals.

FIG. 4B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-0 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997).

At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 4 evenly spaced leucine residues, each separated by 7 other amino acids.

The third domain contains conserved cysteine residues, involved in disulphate bridge formation.

The fourth domain contains a leucine rich repeat domain, consisting of 5 complete repeats of each approximately 24 amino acid residues.

The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and is a site for O-glycosylation.

The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned.

The seventh domain has an unknown function.

The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions.

The ninth domain has an unknown function.

The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 5

Proliferated cell mass of Arabidopsis plants transformed with different overexpressing constructs of RKS genes (A and B) or with a control pGREEN1K vector without RKS genes. After 18 days of proliferation in the presence of 2,4-D, tissues have been grown for 4 weeks in the absence of hormones. Regenerated plantlets and green shoots are clearly visible in transformed tissues A and B, but absent in the control tissues transformed with the empty pGREEN vector (C).

FIG. 6A

Ballistic bombardment of Nicotiana tabacum leaf discs with GT-W-20S at day 0 is followed by a two weeks submerged culture in liquid MS medium 1 mg/L kinetin. Subsequently the discs are cultured on MS agar plates without hormones. Control experiments with empty vector never gave rise to proliferation. The formation of regenerating from leaf explants is shown in days after bombardment.

FIG. 6B

Ballistic bombardment of Nicotiana tabacum leaf discs with GT-SBP5-16S at day 0 is followed by a two weeks submerged culture in liquid MS medium with 1 mg/L kinetin. Subsequently the leaf discs are cultured on MS agar plates without hormones. The formation of regenerating tissues from leaf explants is shown in days after bombardment. Control experiments with empty vectors never gave rise to shoot formation.

FIG. 6C

Nicotiana tabacum callus is bombarded with GT-SBP5-16S at day 0. Callus was generated by incubating tobacco leaves for 6 weeks on MS30, 0.8% agar supplemented with 1 mg/L 2,4-D auxin. The callus that formed on the leaves with root like characteristics (extending roots or root hairs from calli) was further cultured on MS30, 0.8% agar petri dishes. The incubation are performed at 20° C. with 16 hours light, 8 hours dark. Control experiments with empty vectors never gave rise to shoot formation. 40 days after bombardment regenerating plant can be identified on top of the bombarded callus tissue (plant 1 and plant 2).

FIG. 6D

In order to examine the presence of the bombarded DNA regeneration constructs in regenerated plant, tissue samples were taken from 10 different regenerates from the experiments described in the legends of FIG. 6A-C. Genomic DNA was isolated from all samples, as well as from two control plants. On this DNA a PRC reaction was performed using primers specific for the NptII gene: construct 1 and 3 from experiment I.

Oligo's Used for NptII Specific Amplification:

Forward oligo: 5′-GCCATGGTGAACAAGATGGATGG-3′ Reverse oligo: 5′-GGATCCTCAGAAGAACTCGTCAAG-3′. The resulting PCR product was analysed on agarose gel. Lane 1 and 2 represent regenerates from FIG. 6C; Lane 3-6 represent regenerates from FIG. 6A; Lane 7-10 represent regenerates from FIG. 6B. These 10 plants from which tissue material was isolated for lane 1-10 are shown below just prior to DNA isolation. Lane 11 represents a positive control plant that is stable transformed with a control vector (pG1K-GEP). Lane 12 represents a negative control, an untransformed wildtype NTSR1 plant. Lane 13 and 14 represent positive control E. coli purified DNA used for PCR analysis and M represent marker DNA. Results indicate that only the regenerated plant from lane 8 contained a stable integrated NptII sequence, with all controls giving vector DNA bands.

FIG. 6E

Arabidopsis thaliana WS seedlings grown for 14 days on MS agar plates have bombarded with DNA coated gold particles at day 0. Plants are further incubated on the plates at 20° C. with 16 hours light, 8 hours dark. Gold particles were coated with 18 microgram of the construct GT-RKS13. In the bombardment procedure, a GUS expression vector was co-bombarded in combination with the GT-W-20S construct in a molar ration of 10% (GUS versus GT-RKS13). Prior to photography, GUS staining was performed on the bombarded tissues. Cell proliferation (arrow) is detectable on the surface of rosette leaves. Control experiments performed with empty vectors did never result in proliferating tissues.

FIG. 6F

Ballistic bombardment of Arabidopsis thaliana with GT-W-20S constructs results in cell proliferation on top of the rosette leaver (left).

Structures with the morphologic characteristics of somatic embryos appear on the callused structures (middle and right, white arrows). In the bombardment procedure, a GUS expression vector was co-bombarded in combination with the GT-W-20S construct in a molar ration of 10% (GUS versus GT-W-20S). The GT-W-20S construct induces cellular proliferation in neighbouring cells and is unable to induce not contain fragments of the introduced regeneration construct or the GUS expression construct. However, after GUS staining, one cell at the basis of the proliferating cell mass is clearly GUS positive (middle and right, black arrow), indicating that this basal cell has been transformed construct results in the formation of a GUS-negative proliferating cell mass on top of a basal GUS-positive cell. Bombardment studies with empty control vectors did never result in cellular proliferation.

FIG. 6G

Ballistic bombardment of Arabidopsis thaliana Ws with GT-CUC2-S, GT-KNAT1-S and GT-CYCD3-S. Cell proliferation becomes already clearly detectable within one week after bombardment (arrow). Control bombardment studies with empty vectors did not result in cellular proliferation.

FIG. 6H

Ballistic bombardment of Arabidopsis thaliana Ws with GT-CUC-2S, GT-KNAT2-S and GT-CYCD3-3S. Different regions of cell proliferation within individual rosette leaves become already clearly detectable within one week after bombardment (arrows). Control bombardment studies with empty vectors did not result in cellular proliferation.

FIG. 7

The three different RKS subfamilies I-III based on FIG. 3. The predicted protein products are shown, and alignment is based on predicted domain structures. Conserved cysteine residues in disulphate bridge formation are underlined.

From the N-terminus towards the C-terminus these domains can be defined as the signal sequence, the extracellular region consisting of respectively a leucine zipper domain, a disulphate bridge domain, an leucine rich repeat domain with 3-5 leucine rich repeats, a putative hydroxyproline domain involved in O-glycosylation, a single transmembrane domain, an intracellular region consisting of respectively an anchor domain, a serine/threonine kinase domain, a domain with unknown function and at the C-terminus a sequence resembling an intracellular leucine rich repeat.

FIG. 8A

Arabidopsis thaliana RKS1 cDNA

The start codon has been indicated by bold capitals.

FIG. 8B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-1 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 3 leucine residues, each separated by 7 other amino acids. The third domain contains conserved cysteine residues, involved in disulphate bridge formation.

The fourth domain contains a leucine rich repeat domain, consisting of 3 complete repeats of each approximately 24 amino acid residues.

The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation.

The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned.

The seventh domain has an unknown function.

The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions.

The ninth domain has an unknown function.

The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 9A

Arabidopsis thaliana RKS2 cDNA. The start codon has been indicated by bold capitals.

FIG. 9B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-14 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 2 leucine residues, each separated by 7 other amino acids. The third domain contains conserved cysteine residues, involved in disulphate bridge formation.

The fourth domain contains a leucine rich repeat domain, consisting of 4 complete repeats of each approximately 24 amino acid residues. The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation. The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned. The seventh domain has an unknown function. The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions. The ninth domain has an unknown function. The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 10A

Arabidopsis thaliana RKS3 cDNA. The start codon has been indicated by bold capitals.

FIG. 10B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-3 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 3 leucine evenly residues, each separated by 7 other amino acids. The third domain contains conserved cysteine residues, involved in disulphate bridge formation. The fourth domain contains a leucine rich repeat domain, consisting of 4 complete repeats of each approximately 24 amino acid residues. The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation. The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned. The seventh domain has an unknown function. The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions. The ninth domain has an unknown function. The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 11A

Arabidopsis thaliana RKS4 cDNA

The start codon has been indicated by bold capitals.

FIG. 11B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-4 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 2 leucine residues, each separated by 7 other amino acids. The third domain contains conserved cysteine residues, involved in disulphate bridge formation.

The fourth domain contains a leucine rich repeat domain, consisting of 5 complete repeats of each approximately 24 amino acid residues. The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation. The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned. The seventh domain has an unknown function. The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions. The ninth domain has an unknown function. The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 12A

Arabidopsis thaliana RKS5 cDNA. The start codon has been indicated by bold capitals.

FIG. 12B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-5 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 2 leucine residues, each separated by 7 other amino acids. The third domain contains conserved cysteine residues, involved in disulphate bridge formation.

The fourth domain contains a leucine rich repeat domain, consisting of 4 complete repeats of each approximately 24 amino acid residues. The fifth domain has no clear function. The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned. The seventh domain has an unknown function. The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions. The ninth domain has an unknown function. The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 13A

Arabidopsis thaliana RKS6 cDNA. The start codon has been indicated by bold capitals.

FIG. 13B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-6 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 3 leucine residues, each separated by 7 other amino acids. The third domain contains conserved cysteine residues, involved in disulphate bridge formation.

The fourth domain contains a leucine rich repeat domain, consisting of 5 complete repeats of each approximately 24 amino acid residues. The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation. The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned.

The seventh domain has an unknown function. The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions.

The ninth domain has an unknown function.

The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 14A

Arabidopsis thaliana RKS8 cDNA.

The start codon has been indicated by bold capitals.

FIG. 14B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-8 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 4 leucine evenly spaced residues, each seperated by 7 other amino acids. The third domain contains conserved cysteine residues, involved in disulphate bridge formation.

The fourth domain contains a leucine rich repeat domain, consisting of 5 complete repeats of each approximately 24 amino acid residues. The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation.

The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned. The seventh domain has an unknown function.

The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions.

The ninth domain has an unknown function. The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 15A

Arabidopsis thaliana RKS10 cDNA. The start codon has been indicated by bold capitals.

FIG. 15B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-10 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 4 leucine residues, each separated by 7 other amino acids. The third domain contains conserved cysteine residues, involved in disulphate bridge formation.

The fourth domain contains a leucine rich repeat domain, consisting of 4 complete repeats of each approximately 24 amino acid residues. The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation. The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned.

The seventh domain has an unknown function.

The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions.

The ninth domain has an unknown function.

The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 16A

Arabidopsis thaliana RKS11 cDNA/. The start codon has been indicated by bold capitals.

FIG. 16B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-11 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence. The second domain contains a leucine zipper motif, containing 3 leucine residues, each separated by 7 other amino acids.

The third domain contains conserved cysteine residues, involved in disulphate bridge formation. The fourth domain contains a leucine rich repeat domain, consisting of 3 complete repeats of each approximately 24 amino acid residues.

The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation.

The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned. The seventh domain has an unknown function.

The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions.

The ninth domain has an unknown function. The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 17A

Arabidopsis thaliana RKS12 cDNA. The start codon has been indicated by bold capitals.

FIG. 17B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-12 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 2 leucine residues, each separated by 7 other amino acids. The third domain contains conserved cysteine residues, involved in disulphate bridge formation.

The fourth domain contains a leucine rich repeat domain, consisting of 4 complete repeats of each approximately 24 amino acid residues. The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation. The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned.

The seventh domain has an unknown function.

The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions.

The ninth domain has an unknown function.

The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 18A

Arabidopsis thaliana RKS13 cDNA. The start codon has been indicated by bold capitals.

FIG. 18B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-13 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence.

The second domain contains a leucine zipper motif, containing 4 leucine residues, each separated by 7 other amino acids. The third domain contains conserved cysteine residues, involved in disulphate bridge formation.

The fourth domain contains a leucine rich repeat domain, consisting of 4 complete repeats of each approximately 24 amino acid residues. The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation. The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned. The seventh domain has an unknown function. The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions. The ninth domain has an unknown function. The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 19A

Arabidopsis thaliana RKS14 cDNA. The start codon has been indicated by bold capitals.

FIG. 19B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-14 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). At the predicted extracellular domain the first domain represents a signal sequence. The second domain contains a leucine zipper motif, containing 2 leucine residues, each separated by 7 other amino acids.

The third domain contains conserved cysteine residues, involved in disulphate bridge formation. The fourth domain contains a leucine rich repeat domain, consisting of 4 complete repeats of each approximately 24 amino acid residues.

The fifth domain contains many serine and proline residues, and is likely to contain hydroxy-proline residues, and to be a site for O-glycosylation.

The sixth domain contains a single transmembrane domain after which the predicted intracellular domains are positioned. The seventh domain has an unknown function.

The eight domain represents a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions.

The ninth domain has an unknown function. The last and tenth domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 20 A

Arabidopsis thaliana RKS 7 partial cDNA sequence.

The 5′-end and a region between the two cDNA fragments ( . . . ) is not shown.

FIG. 20B

Predicted partial amino acid sequences of the Arabidopsis thaliana RKS-7 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as described in Schmidt et al. (1997). The protein sequence is obtained from partial cDNA sequences. The first available domain represents part of a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions. The next domain has an unknown function. The last domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 21A

Arabidopsis thaliana RKS 9 partial cDNA sequence.

The 5′-end is not shown.

FIG. 21B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-9 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as descibed in Schmidt et al. (1997). The protein sequence is obtained from partial cDNA sequences. The first available domain represents part of a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions. The next domain has an unknown function. The last domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 22A

Arabidopsis thaliana RKS15 partial cDNA sequence.

The 5′-end is not shown.

FIG. 22B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-15 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as descibed in Schmidt et al. (1997). The protein sequence is obtained from partial cDNA sequences. The first available domain represents part of a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions. The next domain has an unknown function. The last domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions.

FIG. 23A

Arabidopsis thaliana RKS16 partial cDNA sequence.

The 5′-end is not shown.

FIG. 23B

Predicted amino acid sequence of the Arabidopsis thaliana RKS-16 protein. Different domains are spaced and shown from the N-terminus towards the C-terminus. Overall domain structure is similar as descibed in Schmidt et al. (1997). The protein sequence is obtained from partial cDNA sequences. The first available domain represents part of a serine/threonine protein kinase domain (Schmidt et al. 1997), and is probably also containing sequences for protein, protein interactions. The next domain has an unknown function. The last domain at the C-terminal end represents a single leucine rich repeat, probably involved in protein, protein interactions. 

1-31. (canceled)
 32. A method for propagation of a plant from non-embryogenic plant starting material, the method comprising stimulating root and/or shoot initiation of said plant starting material by having at least one recombinant gene product or functional fragment thereof transiently present in said starting material, wherein a reduction of exogenous phytohormone to said culture as compared with conventional methods is achieved.
 33. A method for propagation of a plant from non-embryogenic plant starting material, the method comprising stimulating root and/or shoot initiation of said plant starting material by having at least one recombinant gene product or functional fragment thereof transiently present in said starting material, and wherein no exogenous phytohormone is added to said culture.
 34. A method according to claim 32 further comprising transforming a least part of said starting material with a nucleic acid encoding said gene product.
 35. A method according to claim 32 wherein said culture comprises an in vitro culture.
 36. A method according to claim 32 wherein said propagation comprises essentially seedless propagation.
 37. A method according to claim 32 wherein said starting material comprises an individual plant cell, protoplast, explant or plant tissue.
 38. A plant or plant material obtainable by a method according to claim
 32. 